Abstract Dense mammary tissues are 4-5 times more likely to generate tumors than non-dense tissues, such that up to a third of all breast cancers originate in mammographically dense tissue. Increased breast density arises from a significant increase in deposition of extracellular matrix (ECM) constituents, in particular, collagen and fibronectin (FN). Recent studies in murine models have shown that increased collagen density promotes mammary tumor formation and progression. FN is a serum and extracellular matrix glycoprotein whose deposition into fibrillar structures precedes and controls collagen fibril formation in a variety of systems studied. Deposition of fibronectin and contributes to inflammation and fibrosis by recruitment of immune and inflammatory cells that produce pro-fibrotic cytokines. Fibronectin is also a critical component of neo- vascularization, an important component in tumor growth. In contrast to collagen, which can self-assemble, FN fibrillogenesis is tightly controlled by molecules at cell surfaces. We postulate that FN assembly is potentially a drug target and may create an opportunity to regulate subsequent collagen deposition. We hypothesize that inhibitors of FN matrix assembly will diminish fibrosis observed during breast carcinoma progression, and will decrease tumor progression. Our objective is to identify small molecule inhibitors of FN assembly that may be utilized as therapeutics to decrease breast carcinoma incidence, tumorigenicity, and/or metastasis. Two types of inhibitors will be tested. The first type of inhibitor is a peptide derived from Streptococcus pyogenes, termed FUD for Functional Upstream Domain, which we have characterized extensively as a potent and specific inhibitor of FN assembly. Others have utilized FUD in a murine model of cardiovascular remodeling where it inhibited FN and collagen assembly, leukocyte infiltration and inflammation at the injured site. The second type of inhibitors are small molecules identified through a pilot high throughput screen using a FN assembly assay we developed. We chose three of the resulting inhibitors to test in collagen-dense murine models of mammary carcinoma and propose to characterize and test novel inhibitors resulting from a larger screen to take place at the Broad Institute (Cambridge, MA). Two Specific Aims are proposed: 1) Determine if FN deposition affects tumorigenicity of collagen-dense breast carcinomas in mice; 2) Identify and characterize new small molecules that disrupt FN fibrillogenesis derived from High Throughput Screening at the Broad Institute for their efficacy in blocking fibrosis and tumor progression in mouse mammary carcinoma models. The proposed work is novel and significant in that, even though it is well established that collagen alignment and fibrosis accompany tumor progression, the control of collagen and extracellular matrix deposition has not been identified as druggable in the treatment of tumors. Our group combines the needed expertise in collagen dense mammary carcinoma animal models, access to small molecule screening, and state-of-the art intravital imaging technology to identify relevant probes to be potentially developed into therapeutics against breast cancer.